Phthiocerol dimycocerosates promote access to the cytosol and intracellular burden of Mycobacterium tuberculosis in lymphatic endothelial cells

Abstract

BackgroundPhthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs.ResultsAnalysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden.ConclusionsPDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.

Mycobacterium tuberculosis cording in the cytosol of live lymphatic endothelial cells

“…We next sought to understand M. tuberculosis factors that contributed to the intracellular cording phenotype in hLEC. We have previously shown that the ESX-1 secretion system, encoded in the RD1 genomic region, and the cell wall lipid phthiocerol dimycocerosate (PDIMs) are required for intracellular replication of M. tuberculosis in hLEC (Lerner et al, 2016; Lerner et al, 2018). Infection with the M. tuberculosis ΔRD1 mutant that lacks the ESX-1 secretion system was not able to form cords but instead exhibited smaller clumps of bacteria sometimes with a mesh-like appearance ( Fig.…”

“…Because M. tuberculosis intracellular cords formed in the cytosol and induced a pathogen cytosolic recognition signature in hLEC associated with xenophagy (Watson et al, 2012a), we investigated whether M. tuberculosis cords were targeted by selective autophagy. In hLEC, M. tuberculosis targeting via selective autophagy is PDIM dependent (Lerner et al, 2018) and entirely RD1 dependent ( Supplementary Fig. 2 ) suggesting that in hLECs, xenophagy primarily recognises mycobacteria that access the cytosol, the intracellular location for M. tuberculosis cording.…”

“…This study used the following EGFP tagged strains as described previously (Astarie-Dequeker et al, 2009; Lerner et al, 2016; Lerner et al, 2018): Mycobacterium tuberculosis H37Rv-EGFP ( M. tuberculosis WT), M. tuberculosis H37Rv-EGFP ΔRD1 ( M. tuberculosis ΔRD1), M. tuberculosis H37Rv-EGFP ΔRD1::RD1 ( M. tuberculosis ΔRD1::RD1). In this study, we refer to the M. tuberculosis -GFP WT strain as M. tuberculosis WT, the M. tuberculosis -GFP PMM100 strain as M. tuberculosis ΔPDIM.…”

Mycobacterium marinumphthiocerol dimycocerosates enhance macrophage phagosomal permeabilization and membrane damage

“…ESX-1-mediated phagosomal permeabilization is associated with membrane damage in both M. tuberculosis and M. marinum [16,17,27,28,30–32]. M. tuberculosis strains deficient in ESX-1 or PDIM synthesis show reduced galectin-3 or galectin-8 recruitment to intracellular sites of infection[23,24,33,34]. If the endosomal membrane is damaged, cytosolic galectins bind to lumenal β-galactoside-containing glycans that become exposed on damaged vesicles, and can be visualized by immunofluorescence microscopy[23,24,33–36].…”

“…M. tuberculosis strains deficient in ESX-1 or PDIM synthesis show reduced galectin-3 or galectin-8 recruitment to intracellular sites of infection[23,24,33,34]. If the endosomal membrane is damaged, cytosolic galectins bind to lumenal β-galactoside-containing glycans that become exposed on damaged vesicles, and can be visualized by immunofluorescence microscopy[23,24,33–36]. Differing from the role of ESX-1 in phagosomal permeabilization during M. tuberculosis and M. marinum infection, the involvement of M. marinum PDIM in this process has not been demonstrated.…”